B I O T E C H N O L O G Y L E T T E R S Volume 18 No. 11 (November1996) pp.1345-1350 Received as revised 23 September

STABILITY OF IMMOBILIZED ct-CHYMOTRYPSIN IN SUPERCRITICAL CARBON DIOXIDE

Pedro Lozano, Antonio Avellaneda, Raphael Pascual and Jose L. Iborra *

Departamento de Bioquimica y Biologia Molecular B e Inmunologia. Facultad de Quimica. Universidad de Murcia. P.O. Box. 4021. E-30001. Murcia. Spain.

SUMMARY

Inactivation of immobilized c~-chymotrypsin in supercritical carbon dioxide was with a first-order kinetic behaviour. The increase in either the pressure or the temperature of the fluid enhanced the inactivation process of the enzyme. The fluid density was shown as a key parameter on the enzyme stability, enhancing the half-life time proportionally to the physical phase of CO2, as follows: liquid > supercritical > gas. However, the number of pressurization/depressurization cycles, and the water content of the derivative increased greatly the loss of activity.

INTRODUCTION

Enzymatic catalysis in organic solvents can offer many advantages over using pure water (Dordick, 1992; Gupta, 1992). The use of biocatalysts in supercritical fluids, as new non-conventional media, has attracted much interest in last years to create synthetic process by using hydrolytic enzymes (Marty et al., 1990: Chulalaksananukul et al., 1993; Jarzebski and Malinowski, 1995; Noritomi et al., 1995). The physical properties of supercritical fluids, which include gas-like diffusivities and liquid-like densities, can be controlled predictably by changing either the pressure or the temperature, to tune the reaction conditions (Nakamura, 1990; Kamat et al.. 1993). Supercritical carbon dioxide is mainly used as the standard supercritical fluid due to its relatively low critical parameters (P~=72.8 bar, T~=31.5 °C and pc = 0.47 ~mL) and advantages as reaction media over the conventional organic solvents, such as, high mass transfer and diffusion rates, non toxicity, non flammability or low economic cost (Erickson et al., 1990: Aaltonen and Rantakyl~i, 1991; Vermue et al.. 1992). With a supercritical phase system, it is important to consider how the different environmental parameters, such as the pressure, the temperature or the water content of the fluid (which modify the physical properties of the solvent) can change the catalytic behaviour of an enzyme. These facts could result in a severe distortion of the protein structure involving their catalytic deactivation (Chulalaksananukul et aL. 1992; Zagrobelny and Bright, 1992). As a general rule, a detailed understanding of environment-structure-function relationships for proteins in non-conventional media may enable predictive control of enzyme function merely by solvent engineering (Gupta, 1992; Kamat et al., 1993, Lozano eta/.. 1995, 1996). However, no systematic study on the enzyme stability under supercritical conditions has yet been carried out. The analvsis of enzyme deactivation, and the possibility of enhancing its stability, are important considerations to develop biocatalytic process in supercritical systems. In this work, the influence of pressure and temperature on supercritical carbon dioxide on the stability of an immobilized ct-chymotrypsin has been studied. Additionally, the effect of other

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environmental conditions, as the water content of the immobilized derivative, as well as the number of consecutive pressurization-depressurization cycles has also been analyzed.

MATERIALS AND METHODS Materials o~-Chymotrypsin (EC 3.4.21.1., type II from porcine pancreas), and N-acetyl-L-tyrosine ethyl ester were from Sigma, and used without previous purification. Celite 545 (0.01 - 0.04 mm particle size) were obtained from Merck. Carbon dioxide (Purity 99.85%, water content 0.005%) was purchased from Argon (Praxair Group, Spain). Immobilization process. ~-Chymotrypsin (250 rag) was dissolved in 30 mL O. 1 M phosphate buffer pH 7.0, mixed with 5 g Celite and shaken for 2 h at room temperature and then freeze dried. The amount of protein adsorbed on the support was 95%, measured by the Lowry's method. The immobilized derivative showed an esterase activity towards N-acetyl-L-tyrosine ethyl ester of 15.73 U/mg support (Immobilization Yield = 30%) and a water-content of 0.1 mg H20/g support, determined by Karl- Fischer method. Study of enzyme stability in supercritical CO2. Immobilized ct-chymotrypsin derivative (150 mg) was packed into a plastic cylinder, having a porous membrane of nylon (0.5 mm pore size) at the extremes, to allow the free contact of the supercritical fluid with the immobilized derivative particles. The plastic vessel was then placed into the high pressure reactor and incubated at different supercritical conditions, determined by changes in pressure and temperature of the system. As it can be seen in Figure 1, liquid carbon dioxide was pumped, by using a membrane pump (Milroyal B, Milton Roy, France), through the steel tubular reactor (R), being previously heated at the assayed temperature by the exchanger heater (H) (see Figure 1).

R: Reactor H: Heater C: Cooler HPP: High Pressure Pump P: Pressure Control T: Temperature Control Vt, V=: Valves SV: Security Valve

H C

C

o t.j

-1

m m

Figure 1. Experimental set-up of the supercritical fluids reactor

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The opening/closing grade of the needle valve V2 (Whitey SS-31RS4, Ohio) establishes the pressure into the system to give the supercritical conditions, being controlled by the pressure display, P, and the excess of fluid was recycled. When the pressure into the system was stabilized, the valves Vt and Vz were closed and then, the system was maintained until opening to measure the residual activity. Assay of enzyme activity. The esterase activity ofct-chymotrypsin was determined by the pH-stat method described in detail by Wilcox (1970). A video-titrator VIT-90 equipped with an autoburette (ABU 91) and a sample station SAM 90 (Radiometer, Copenhagen) were used. In brief the protocol was as follows: a 3 mL sample of 50 mM ATEE in 30% (v/v) ethanol containing 20 mM CaC12 was placed into a thermostated (40 °C) reaction vessel. The reaction was started by addition of 50 ~.tL 0.1 (w/v) aqueous suspension of the immobilized a-chymotrypsin solution previously homogenized. The pH was maintained constant at 7.0 by continuous addition of 50 mM NaOH as titrant. One unit of activity was defined as the amount of enzyme that hydrolyzes one micromole of ATEE per minute under the standard conditions of assay (pH 7.0, 40 °C).

RESULTS AND DISCUSSION Influence of pressure and temperature of carbon dioxide on enzyme stability. The effect of different physical states of carbon dioxide, determined by changes in pressure and temperature of the fluid, on the immobilized c~-chymotrypsin stability was studied in anhydrous media. Figure 2 shows the deactivation profiles of the immobilized derivative at the assayed supercritical conditions. The assayed pressure and temperature were chosen as a function of the phase diagram of CO~, in order to have different conditions in supercritical phase (80 and 150 bar at 40 °C, and 100 and 150 bar at 60 °C), as well as in liquid (100 bar at 25 °C) and gas (55 bar at 40 °C and 60 bar at 60 °C) phases near to supercritical phase. In all cases, the experimental results showed an activity decay corresponding to a one-step first-order model. As it can be seen in Figure 2, the

1.95

1.90

"~ 1.80

"~ 2.00

8' 1.95 , - I

1.90

1.85

I I I I I

1 . 8 0 I I I I I 0 5 10 15 20 25 30

T i m e ( h )

Figure 2: Deactwation profiles of immobilized cx-chymot .rypsin in carbon dioxide at different conditions: Liquid [(o) 100 bar. 25 °C]; Supercritical [(!1,) 150 bar. 40 °C: (&) 80 bar, 40 °C: (l"l) 150 bar. 60 "C: (A) 100 bar. 60 °C]; Gas: [(ll) 55 bar. 40 °C: (O) 60 bar. 60 °C].

immobilized c~-chymotrypsin stability was sensitive to both, the pressure and the temperature experimental conditions. In liquid-near critical phase (100 bar, 25 °C), the enzyme activity was remained unchanged with time, showing a 100% of enzyme activity after three days of incubation without any deactivation process. In supercritical phase, the increase in pressure produced an enhancement of the enzyme stability at each assayed temperature (40 and 60 °C). However, the enzyme activity was clearly reduced by the increase in temperature, mainly due to the thermal denaturation of the enzyme (Chulalaksananukul et al., 1993). On the other hand, in gas-near critical phase (55 bar, 40 °C and 60 bar, 60 °C) the enzyme derivative showed a clear activity decay with time. The attained effect of temperature, as

1347

unstabilizing parameter, contrasts with the stabilizing effect of pressure. At high pressures, activity of some enzymes decreases due to denaturation; however, the pressure of supercritical carbon dioxide is not excessively high and its direct effect with regard to protein unfolding should be small (Nakamura, 1990; Kamat et al., 1993). Thereby, to unify the influence of supercritical media on the enzyme stability, the density parameter of the fluid has been chosen (Kennedy and 40 Thodos, 1960). Figure 3 shows the evolution of the half-life time of the immobilized ct-chymotrypsin as a ~, 30 function of density of carbon dioxide, at "~ the different assayed gas-near-critical and supercritical conditions, derivative '= ~ 20 (liquid-near critical half-life time can not be calculated). As it can be seen, the 10 enzyme half-life time was proportionally enhanced by the increase in the density of the fluid. These results allow to show 0 the density of carbon dioxide as a 0.0 1.0 protective parameter towards enzyme deactivation, allowing to order the physical phase of fluid, as follows:

Figure 3: Effect of density of carbon dioxide on the half-life time liquid > supercriticat > gas. It is also of immobilized c~_chynlott3.psin necessary to mention that this protective effect of supercritical media can attain an important value: the stability of the immobilized derivative was increased in 200-times (for 150 bar, 40 °C) with respect to the aqueous media, and 14-times respect to a stabilizing hydrophilic-organic media, as a 30% (v/v) aqueous dimethyl sulphoxide solution (Lozano et aL, 1996). These results should be explained as a function of the changes in physical properties of carbon dioxide. The increase in density of carbon dioxide produces the enhancement of several parameters as, the dielectric constant, the polarity/polarizability or the Hildebrand solubility of the solvent, which involves the increase in the hydrophilic character of the solvent (Smith et al., 1987; Vermue et al., 1992; Kamat et al., 1993). Thus, in the same way of organic media, the increase of solvent hydrophilicity allowed to enhance the enzyme stability by increase in solvent-protein interactions with the polar groups of the enzyme, preserving the solvophobic interactions into the protein, which play a key role in supporting the active conformation (Mozhaev et al., 1989; Lozano et al.,

1996)

V/" I I I I

0.2 0.4 0.6 0.8 D e n s i t y ( g / L )

Effect of pressurization/depressurization cycles. The effect of pressurization/depressurization steps on the enzyme stability was also studied. Figure 4 shows the influence of consecutive cycles of pressurization/depressurization steps on the evolution of the residual activity of immobilized ct-chymotrypsin, using 150 bar and 40"C, as standard supercritical conditions. In each cycle, the pressurizatiorv'depressurization steps were made for 15 min, while the incubation period for the immobilized derivative was during 1 h. Considering the low effect of these supercritical incubation conditions on the enzyme deactivation for 1 h (Figure 2), it

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can be observed how there was a considerable influence of the pressurization / depressurization cycles on the enzyme stability, reducing the activity profile. Thus, the pressurization/depressurization steps in each cycle were shown as a high deactivation parameter, producing a continuous activity decay, which was higher than 30°,/0 after 9 cycles, without any loss in protein concentration on the immobilized derivative. Several authors (Kasche et al., 1988, Nakamura, 1990; Aaltonen and Rantakyla, 1991) have described the influence of the pressurization/depressurization steps on the enzyme stability. Kasche et al.

(1988) suggested that enzyme deactivation by depressurization is probably due to a structural change in the protein molecule caused by the decrease in pressure on rapid release of CO2 dissolved in the bound water. Zagrobelny and Bright (1992) have made m situ studies of trypsin conformation changes in supercritical carbon dioxide by fluorescence spectroscopy. However, these authors concluded that it is the pressurization step that produces conformational changes in the protein structure, where the protein unfolds to a denatured state, but the depressurization step has very little effect.

A

100

, I

._> 75

- - 5 0

"~ 25 r~

0

- 0 0 0 0 0 0

0 0 0

0 2 4 6 8 10 P r e s s u r i z a t i o n I D e p r e s s u r i z a t i o n

Cycles

Figure 4. Effect of pressurizatioxVdepressurization cycles on the residual activit3.' of immobilized c~-chymotrypsin in supercritical carbon dioxide (150 bar and 40 °C).

Effect o f w a t e r content . In nonaqueous environment or low-water media, enzymes can function provided if the essential water layer around them is not stripped off (Dordick, 1992). Not surprisingly, all studies published so far pointed out the strong influence of moisture on enzymic activity and reaction rates. In this way, the c~-chymotrypsin stability on supercritical carbon dioxide (150 bar and 40 °C) was also analyzed as a function of the water content of the immobilized derivative. As it can be seen in Table I, the increase in the amount of water added to the immobilized enzyme produced an exponential decrease in the half-life time of the enzyme activity. It is also important to notice how at the highest assayed water

Table I. Effect of the added water amount ou stabilib' of ilnmobilized ~-chynmtrypsin in supercritical phase (150 bar and 40°C)

Added Water Amount

% (w/w)

0.0

2.8

4.5

8.0

15.0

Half-life time

(h)

770.2

61.0

8.1

5.0

3.5

addition, the half-life time of the immobilized derivative was identical to the observed previously in 100% aqueous media (Lozano et al., 1996). Supercritical carbon dioxide may dissolve as much as 0.3 - 0.35% water (Jarzebski and Malinowski, 1995) and, at the end of all experiments it was observed that the moisture of the support was remained practically unchanged. So, the protective effect of the supercritical solvent was clearly reduced by exclusion of carbon dioxide molecules from the domain of protein molecules. These results are in agreement with other authors (Vermue et aL,

1349

1992; Chulalaksananukul et al., 1993), which shows the negative influence of the water on the enzyme activity and stability in supercritical media. As function of these results, the supercritical carbon dioxide could also be classified as hydrophobic solvent, in terms of the low protective effect on enzyme stability: the higher solvent hydrophobicity, the stronger denaturative effect on proteins (Gupta, 1992; Lozano et al., 1996). Therefore, the optimum water content for an enzymic activity in supercritical carbon dioxide media should be a compromise between the activity and the stability of the enzyme, where the presence of hydrophilic cosolvents (entrainers) could improve the biocatalytic actions.

A C K N O W L E D G E M E N T S .

This work was partially supported by the grants from "Comunidad Aut6noma de la Region de Murcia n ° PTC 93/27", and "Comision Interministerial de Ciencia y Tecnologia, CICYT n ° BIO96- 1016-C02-01 ". Spain. R. Pascual was a fellow of the EEC Erasmus Programme, from Universite de Picardie-Jules Verne. Amiens. France.

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698. Dordick, J.S. (1992). BJotechnol Progr., 8, 259 - 267. Erickson, J.C., Schyns, P. and Cooney, C.L. (1990). AIChEJ., 36, 299-301. Gupta, M.N (1992). Eur. J. Biochem., 203, 25 - 32. Jarzebski, A.B. and Malinowski, J.J. (1995). Plvcess Biochem., 30, 343 - 352. Kamat, S., Iwaskewycz, B., Beckman, E.J. and Russell, A.J. (1993). Proc. Natl. Acad. Sci. 90, 2940

- 2944. Kasche, V.; Schlothauer, R. and Brunner, G. (1988). Biotechnol. Lett., 10, 569 - 574. Kennedy, J.T. and Thodos, G. (1960). J. Chem. Eng. Data. 5, 293-297. Lozano, P., de Diego, T, and Iborra, J.L. (1995). Biotechnol. Left.. 17, 603-608. Lozano, P., de Diego, T, and Iborra, J.L. (1996). Biotechnol. Progr. 12, 488 - 493. Marty, A., Chulalaksananukul., W., Condoret, J.S., Willemont, R.M. and Durand, G. (1990).

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and Martinek, K. (1989). Era'. J. Biochem., 184, 597 - 602 Nakamura, K. (1990). TIBTECH, 8, 288292. Noritomi, H., Miyata, M., Kato, S. and Nagahama, K. (1995). Biotechnol. Lett., 17, 1323 - 1328. Smith, R.S., Frye, S.L., Yonker, C.R. and Gale, R.W. (1987). Phys. Chem., 91, 3059 - 3062. Vermue, M.H., Tramper, J., de Jong, J.P.J. and Oostrom, WH.M. (1992). Enzyme Micro&

Technol., 14, 649 - 655. Wilcox, P.E. (1970). Meth. Enzymol., 19, 64 - 108. Zagrobelny, J. and Bright, F. (1992). Biotechnol. Progr. 8~ 421-423.

1 3 5 0